Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ordered and dynamic assembly of single spliceosomes.

ABSTRACT: The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

SUBMITTER: Hoskins AA

PROVIDER: S-EPMC3086749 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Ordered and dynamic assembly of single spliceosomes.

Hoskins Aaron A AA Friedman Larry J LJ Gallagher Sarah S SS Crawford Daniel J DJ Anderson Eric G EG Wombacher Richard R Ramirez Nicholas N Cornish Virginia W VW Gelles Jeff J Moore Melissa J MJ

Science (New York, N.Y.) 20110301 6022

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is ...[more]

PMID: 21393538

Similar Datasets

Synergistic assembly of human pre-spliceosomes across introns and exons.

Project description:Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.

| S-EPMC6035042 | biostudies-literature

Constitutive nucleosome depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule footprinting.

Project description:Chromatin organization and transcriptional regulation are interrelated processes. A shortcoming of current experimental approaches to these complex events is the lack of methods that can capture the activation process on single promoters. We have recently described a method that combines methyltransferase M.SssI treatment of intact nuclei and bisulfite sequencing allowing the representation of replicas of single promoters in terms of protected and unprotected footprint modules. Here we combine this method with computational analysis to study single molecule dynamics of transcriptional activation in the stress inducible GRP78 promoter. We show that a 350-base pair region upstream of the transcription initiation site is constitutively depleted of nucleosomes, regardless of the induction state of the promoter, providing one of the first examples for such a promoter in mammals. The 350-base pair nucleosome-free region can be dissected into modules, identifying transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress. The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states. We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site. Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes.

| S-EPMC1574359 | biostudies-literature

Self-assembly of ordered graphene nanodot arrays.

Project description:The ability to fabricate nanoscale domains of uniform size in two-dimensional materials could potentially enable new applications in nanoelectronics and the development of innovative metamaterials. However, achieving even minimal control over the growth of two-dimensional lateral heterostructures at such extreme dimensions has proven exceptionally challenging. Here we show the spontaneous formation of ordered arrays of graphene nano-domains (dots), epitaxially embedded in a two-dimensional boron-carbon-nitrogen alloy. These dots exhibit a strikingly uniform size of 1.6?±?0.2?nm and strong ordering, and the array periodicity can be tuned by adjusting the growth conditions. We explain this behaviour with a model incorporating dot-boundary energy, a moiré-modulated substrate interaction and a long-range repulsion between dots. This new two-dimensional material, which theory predicts to be an ordered composite of uniform-size semiconducting graphene quantum dots laterally integrated within a larger-bandgap matrix, holds promise for novel electronic and optoelectronic properties, with a variety of potential device applications.The nanoscale patterning of two-dimensional materials offers the possibility of novel optoelectronic properties; however, it remains challenging. Here, Camilli et al. show the self-assembly of large arrays of highly-uniform graphene dots imbedded in a BCN matrix, enabling novel devices.

| S-EPMC5491516 | biostudies-literature

Hierarchically ordered self-assembly of amphiphilic bifullerenes.

Project description:A series of novel functionalised dumbbell-shaped bifullerenes in which two [5.0] pentakis-adducts of C60 are covalently connected by cyclic bismalonates were synthesised. These dimeric compounds, carrying various combinations of hydrophilic and hydrophobic addends, self-assemble in aqueous solution towards supramolecular architectures of different structural complexity as observed by cryogenic transmission electron microscopy (cryo-TEM). The detailed analysis of the image data revealed an unprecedented hierarchical aggregation behaviour. Whereas completely hydrophilic substituted bifullerenes formed profoundly monodisperse populations of small oligomeric elementary micelles consisting of only three or four bifullerene molecules in a supposedly bent conformation, their amphiphilic equivalents underwent a hierarchical two-step assembly process towards larger spherical and even rod-like structures. The data suggest that the hierarchical assembly process is driven by hydrophobic interactions of preformed tetrameric elementary micelles.

| S-EPMC4744996 | biostudies-literature

Ordered assembly of two different metal clusters with the same topological connectivity in one single coordination network.

Project description:The introduction of heterogeneous components within one single coordination network leads to the multifunctionality of the final material. However, it is hard to precisely control the local distribution of these different components in such a coordination network, especially for different components with identical topological connectivity. In this study, we successfully achieved the ordered assembly of [Mn3(μ3-O)] nodes and [Mn6(μ3-O)2(CH3COO)3] nodes within one pacs coordination network. The resulting new structure (NPU-6) with heterogeneous metal nodes simultaneously inherits the advantages of both parent networks (good thermal stability and high pore volume). The significant effect of the reaction concentration of competing ligand CH3COO- on the mixed assembly of these two nodes in NPU-6 is revealed by a series of control experiments. This method is anticipated to offer a valuable reference for orderly assembling heterogeneous components in coordination networks.

| S-EPMC11290453 | biostudies-literature

Ordered polyelectrolyte assembly at the oil-water interface.

Project description:Polyelectrolytes (PEs) are widely used in applications such as water purification, wastewater treatment, and mineral recovery. Although much has been learned in past decades about the behavior of PEs in bulk aqueous solutions, their molecular behavior at a surface, and particularly an oil-water interface where many of their applications are most relevant, is largely unknown. From these surface spectroscopic and thermodynamics studies we report the unique molecular characteristics that several common polyelectrolytes, poly(acrylic acid) and poly(methylacrylic acid), exhibit when they adsorb at a fluid interface between water and a simple insoluble organic oil. These PEs are found to adsorb to the interface from aqueous solution in a multistepped process with a very thin initial layer of oriented polymer followed by multiple layers of randomly oriented polymer. This additional layering is thwarted when the PE conformation is constrained. The adsorption/desorption process is highly pH dependent and distinctly different than what might be expected from bulk aqueous phase behavior.

| S-EPMC3295288 | biostudies-literature

Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes

Project description:Transcription initiation involves the coordinated activities of large multimeric complexes that are organized into functional modules. Little is known about the mechanisms and pathways that govern their assembly from individual components. We report here several principles governing the assembly of the highly conserved SAGA and NuA4 co-activator complexes. Using fission yeast, which contain two functionally non-redundant paralogs of the shared Tra1 subunit, we demonstrate that Tra1 contributes to scaffolding the entire NuA4 complex. In contrast, within SAGA, Tra1 specifically promotes the incorporation of the de-ubiquitination module (DUB), defining an ordered assembly pathway. Biochemical and functional analyses elucidated the mechanism by which Tra1 assemble differentially into SAGA or NuA4 and identified a small, conserved region of Spt20 that is both necessary and sufficient to anchor Tra1 within SAGA. Finally, we establish that Hsp90 and its cochaperone TTT are required for Tra1 de novo incorporation into both SAGA and NuA4, indicating that Tra1, a pseudokinase of the PIKK family, shares a dedicated chaperone machinery with its cognate kinases. Overall, our work brings mechanistic insights into the de novo assembly of transcriptional complexes through ordered pathways and reveals the contribution of dedicated chaperones to this process.

2019-09-30 | GSE128448 | GEO

Ordered assembly of the duplicating Golgi in Trypanosoma brucei.

Project description:The new Golgi in the protozoan parasite Trypanosoma brucei grows near to the old and adjacent to the growing new endoplasmic reticulum exit site. Growth is now shown to be at least a two-stage process, in which a representative matrix marker (GRASP) and enzyme (GntB) are delivered to the site of assembly, followed approximately 10 min later by a COPI component (epsilon-COP) and a trans-Golgi network (TGN) marker (GRIP70). A secretory cargo marker (signal sequence-YFP) appeared early near the new endoplasmic reticulum exit site but did not enter the Golgi until the second stage. Together these data suggest that structural and enzymatic components of the new Golgi stack are laid down first, followed by those needed to move and sort the cargo passing through it.

| S-EPMC1472504 | biostudies-literature

Membrane binding controls ordered self-assembly of animal septins.

Project description:Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

| S-EPMC8099429 | biostudies-literature

The role of ordered cooperative assembly in biomolecular condensates.

Project description: Not available

| S-EPMC9156444 | biostudies-literature